Clostridium difficile infection (CDI) is a major identifiable cause of nosocomial antibiotic-associated diarrhea. In the US alone, there are approximately 500,000 CDI cases annually, with a mortality rate >2.5%. The annual CDI-associated costs are estimated at $3.2 billion. The infectious form of C. difficile is the spore, a resistant structure that can survive on hospital surfaces for extended periods. C. difficile spore do not cause disease, but can revert to toxin-producing bacteria (a process called germination) in the microbiota-depleted gut of hospitalized patients. We have found a synthetic bile salt (CamSA) that inhibits C. difficile spore germination and more importantly protects mice from CDI without toxic effects. CamSA also synergizes with vancomycin to protect hamsters from CDI. Although CamSA has many attractive properties, it still needs to be optimized for pre-clinical trials. We have assembled a team with complementary expertise to help improve the pharmacological properties of CamSA. At the start of this project, we will finish optimizing the necessary assays using CamSA as our lead compound. The data acquired for CamSA will serve as benchmarks for new analogs (specific aim 1) to be tested in vitro and in vivo (specific aims 2-4). At each step, GO/NO GO criteria will eliminate sub-optimal analogs. The information gathered will be used to inform the rational design and synthesis of more potent and stable anti- germinants. This iterative process will be continued until an optimized CDI prophylactic lead is obtained. The specific aims are designed to be interdependent. CamSA has already moved through much of this process since it was easily synthesized (specific aim 1), tested in a battery of in vitro assays (specific aim 2), and tested as a CDI prophylactic in rodents (specific aim 3). Moreover, we have primed the pump by finding three new anti-germinants that are ready for in vitro and in vivo testing.